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The MHC-II transactivator CIITA inhibits Tat function and HIV-1 replication in human myeloid cells.

Forlani G, Turrini F, Ghezzi S, Tedeschi A, Poli G, Accolla RS, Tosi G - J Transl Med (2016)

Bottom Line: Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells.This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.

ABSTRACT

Background: We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells.

Methods: U937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants.

Results: CIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.

Conclusions: U937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1 replication. The generation of Plus-CIITA cells was instrumental to demonstrate the specific contribution of CIITA in terms of inhibition of Tat activity and HIV-1 restriction, independently from other cellular factors, including TRIM22. Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

No MeSH data available.


Related in: MedlinePlus

Residues 253-285 of CIITA are essential but not sufficient to inhibit Tat-mediated HIV-1 LTR transactivation. a 293T cells were co-transfected with fixed amounts of pHIV-1LTR Luc, phRL-CMV, pTat and increasing amounts of flag-tagged CIITA full-length or the deletion mutants listed below the western blot analyses. The results of a representative experiment are shown. Mean luciferase activities, normalized to Renilla activity, are presented as percentages relative to activation by Tat set to 100 % (black column 2). Column 1 represents the basal activity of cells transfected with the pcDNA3 vector. The expression of recombinant fCIITA proteins in cell extracts was evaluated by anti-flag western blotting. b Schematic representation of the results of the gene reported assay illustrated in panela. CIITA proteins used for the mapping are shown, along with their capacity to inhibit Tat-dependent activation of LTR promoter (+). The endpoints of CIITA full-length and CIITA deletion mutants are indicated. At the top is a diagram of CIITA with its domains, labeled as follows: AD activation domain, P/S/T proline/serine/threonine-rich domain, GBD GTP-binding domain, and LRR leucine-rich repeats
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Fig4: Residues 253-285 of CIITA are essential but not sufficient to inhibit Tat-mediated HIV-1 LTR transactivation. a 293T cells were co-transfected with fixed amounts of pHIV-1LTR Luc, phRL-CMV, pTat and increasing amounts of flag-tagged CIITA full-length or the deletion mutants listed below the western blot analyses. The results of a representative experiment are shown. Mean luciferase activities, normalized to Renilla activity, are presented as percentages relative to activation by Tat set to 100 % (black column 2). Column 1 represents the basal activity of cells transfected with the pcDNA3 vector. The expression of recombinant fCIITA proteins in cell extracts was evaluated by anti-flag western blotting. b Schematic representation of the results of the gene reported assay illustrated in panela. CIITA proteins used for the mapping are shown, along with their capacity to inhibit Tat-dependent activation of LTR promoter (+). The endpoints of CIITA full-length and CIITA deletion mutants are indicated. At the top is a diagram of CIITA with its domains, labeled as follows: AD activation domain, P/S/T proline/serine/threonine-rich domain, GBD GTP-binding domain, and LRR leucine-rich repeats

Mentions: CIITA is a protein composed of 1130 amino acids, containing several functional domains regulating its biological activity. To define the region(s) of CIITA responsible for inhibiting the activation of the HIV-1 promoter by Tat, several deletion mutants of CIITA were expressed in 293T cells and analyzed for their ability to suppress Tat-promoted HIV-1 LTR-dependent luciferase activities in comparison to full-length CIITA. Confirming the results obtained in CIITA-positive U937 cells, CIITA full-length inhibited Tat activity in a dose-dependent manner (Fig. 4a, lanes 3–5 vs lane 2). By using two complementary CIITA fragments, 1-321 and 322–1130, we determined that the N-terminal 1–321, but not the C-terminal 322–1130 deletion mutant, inhibited Tat activity (Fig. 4a, lanes 6–8 and lanes 9–11, respectively). Thus, we focused on the N-terminal part of CIITA to restrict the region mediating its inhibitory effect. The mutant encompassing amino acids 1–285 still inhibited Tat function, similarly to CIITA 1–321 (Fig. 4a, lanes 12–14 vs lane 2). On the contrary, CIITA 1-252 did not exert any suppressive effect (Fig. 4a, lanes 15–17 vs lane 2) indicating that residues 253–285 were crucial for the inhibition of Tat function (Fig. 4b, black box). Surprisingly enough, the CIITA fragment 253–410, containing the inhibitory domain, did not block LTR transactivation by Tat (Fig. 4a, lanes 18–20 vs lane 2), suggesting that the inhibitory domain 253–285 must be extended at the N-terminus in order to accomplish its suppressive effect. Indeed, the extension of fragment 253–410 to residue 1 rescued the ability to inhibit Tat by CIITA 1-410 (Fig. 4a, lanes 21–23 vs lane 2). Of interest, the deletion of the first 63 amino acids in CIITA 1–285 did not abrogate its capacity to suppress Tat activity (Fig. 4a, lanes 24–26 vs lane 2). Overall, these results show that the CIITA region encompassing residues 64–285 is the minimal domain required for the inhibition of Tat-dependent LTR transactivation.Fig. 4


The MHC-II transactivator CIITA inhibits Tat function and HIV-1 replication in human myeloid cells.

Forlani G, Turrini F, Ghezzi S, Tedeschi A, Poli G, Accolla RS, Tosi G - J Transl Med (2016)

Residues 253-285 of CIITA are essential but not sufficient to inhibit Tat-mediated HIV-1 LTR transactivation. a 293T cells were co-transfected with fixed amounts of pHIV-1LTR Luc, phRL-CMV, pTat and increasing amounts of flag-tagged CIITA full-length or the deletion mutants listed below the western blot analyses. The results of a representative experiment are shown. Mean luciferase activities, normalized to Renilla activity, are presented as percentages relative to activation by Tat set to 100 % (black column 2). Column 1 represents the basal activity of cells transfected with the pcDNA3 vector. The expression of recombinant fCIITA proteins in cell extracts was evaluated by anti-flag western blotting. b Schematic representation of the results of the gene reported assay illustrated in panela. CIITA proteins used for the mapping are shown, along with their capacity to inhibit Tat-dependent activation of LTR promoter (+). The endpoints of CIITA full-length and CIITA deletion mutants are indicated. At the top is a diagram of CIITA with its domains, labeled as follows: AD activation domain, P/S/T proline/serine/threonine-rich domain, GBD GTP-binding domain, and LRR leucine-rich repeats
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835826&req=5

Fig4: Residues 253-285 of CIITA are essential but not sufficient to inhibit Tat-mediated HIV-1 LTR transactivation. a 293T cells were co-transfected with fixed amounts of pHIV-1LTR Luc, phRL-CMV, pTat and increasing amounts of flag-tagged CIITA full-length or the deletion mutants listed below the western blot analyses. The results of a representative experiment are shown. Mean luciferase activities, normalized to Renilla activity, are presented as percentages relative to activation by Tat set to 100 % (black column 2). Column 1 represents the basal activity of cells transfected with the pcDNA3 vector. The expression of recombinant fCIITA proteins in cell extracts was evaluated by anti-flag western blotting. b Schematic representation of the results of the gene reported assay illustrated in panela. CIITA proteins used for the mapping are shown, along with their capacity to inhibit Tat-dependent activation of LTR promoter (+). The endpoints of CIITA full-length and CIITA deletion mutants are indicated. At the top is a diagram of CIITA with its domains, labeled as follows: AD activation domain, P/S/T proline/serine/threonine-rich domain, GBD GTP-binding domain, and LRR leucine-rich repeats
Mentions: CIITA is a protein composed of 1130 amino acids, containing several functional domains regulating its biological activity. To define the region(s) of CIITA responsible for inhibiting the activation of the HIV-1 promoter by Tat, several deletion mutants of CIITA were expressed in 293T cells and analyzed for their ability to suppress Tat-promoted HIV-1 LTR-dependent luciferase activities in comparison to full-length CIITA. Confirming the results obtained in CIITA-positive U937 cells, CIITA full-length inhibited Tat activity in a dose-dependent manner (Fig. 4a, lanes 3–5 vs lane 2). By using two complementary CIITA fragments, 1-321 and 322–1130, we determined that the N-terminal 1–321, but not the C-terminal 322–1130 deletion mutant, inhibited Tat activity (Fig. 4a, lanes 6–8 and lanes 9–11, respectively). Thus, we focused on the N-terminal part of CIITA to restrict the region mediating its inhibitory effect. The mutant encompassing amino acids 1–285 still inhibited Tat function, similarly to CIITA 1–321 (Fig. 4a, lanes 12–14 vs lane 2). On the contrary, CIITA 1-252 did not exert any suppressive effect (Fig. 4a, lanes 15–17 vs lane 2) indicating that residues 253–285 were crucial for the inhibition of Tat function (Fig. 4b, black box). Surprisingly enough, the CIITA fragment 253–410, containing the inhibitory domain, did not block LTR transactivation by Tat (Fig. 4a, lanes 18–20 vs lane 2), suggesting that the inhibitory domain 253–285 must be extended at the N-terminus in order to accomplish its suppressive effect. Indeed, the extension of fragment 253–410 to residue 1 rescued the ability to inhibit Tat by CIITA 1-410 (Fig. 4a, lanes 21–23 vs lane 2). Of interest, the deletion of the first 63 amino acids in CIITA 1–285 did not abrogate its capacity to suppress Tat activity (Fig. 4a, lanes 24–26 vs lane 2). Overall, these results show that the CIITA region encompassing residues 64–285 is the minimal domain required for the inhibition of Tat-dependent LTR transactivation.Fig. 4

Bottom Line: Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells.This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.

ABSTRACT

Background: We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells.

Methods: U937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants.

Results: CIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.

Conclusions: U937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1 replication. The generation of Plus-CIITA cells was instrumental to demonstrate the specific contribution of CIITA in terms of inhibition of Tat activity and HIV-1 restriction, independently from other cellular factors, including TRIM22. Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

No MeSH data available.


Related in: MedlinePlus